Project Summary/Abstract ! The mechanistic target of rapamycin complex 1 (mTORC1) is a major regulator of cell growth that is frequently deregulated in human diseases such as cancer, diabetes, and aging1-5. The mTORC1 pathway integrates a diverse set of inputs such as growth factor signaling, energy levels, and nutrient availability in order to coordinate cell anabolism and catabolism with environmental conditions and organismal physiology1,2. Among the inputs upstream of mTORC1, the amino acid leucine has long been known to be of critical importance16. In mammals, leucine stimulates a variety of important physiological phenomena including muscle growth, satiety, and insulin secretion, in large part through activation of mTORC117-19. Amino acids, including leucine, signal to mTORC1 through the Rag GTPases, indirect activators of mTORC111. In addition, the GATOR1 complex functions as a GAP for RagA/B, while GATOR2 interacts with and inhibits GATOR115. Recent IP-MS analyses identified the Sestrins (Sestrin1-3) as GATOR2 interacting proteins that inhibit the mTORC1 pathway in the absence of amino acids20,21. Subsequent biochemical analyses revealed that the Sestrin2-GATOR2 interaction is directly sensitive to leucine in vitro, and Sestrin2 binds leucine with a dissociation constant (KD) of approximately 20 M, which together with cell signaling data helped demonstrate that Sestrin2 in a bona fide leucine sensor for mTORC122. To understand the biochemical mechanism of leucine sensing by Sestrin2, we have solved the crystal structure of leucine-bound Sestrin2 at 2.7, revealing the structural basis for leucine detection by Sestrin223. This analysis revealed an evolutionarily unique leucine binding pocket, as well as the presence of a ?lid-latch? mechanism required for leucine binding. Despite these insights however, the structure raised several more questions regarding the mechanism of leucine sensing by the Sestrins. In this proposal, we aim to address these questions through the following specific aims: 1. Characterize the kinetics of leucine dissociation from Sestrin2. 2. Determine the structural basis for the different leucine binding affinities of Sestrin paralogs 3. Characterize the structure of GATOR2-bound Sestrin2 !